(i) Field of the Invention
The present invention relates to nucleic acid molecules encoding polypeptides having the enzymatic activity of a fructosyltransferase. The invention furthermore relates to vectors and hosts containing such nucleic acid molecules. The present invention also relates to processes for producing fructosyltransferase and the production of polyfructoses of the inulin type in various host organisms or in vitro, as well as the fructosyltransferase encoded by the nucleic acid molecules described which may be used to produce polyfructoses of the inulin type.
(ii) Description of the Related Art
Water-soluble, linear polymers can be put to various uses, for examples for increasing the viscosity of aqueous systems, as detergents, as suspension agents or for sedimentation acceleration and complexing, but also to bind water. Saccharide-based polymers, for example fructosyl polysaccharides, are particularly interesting starting materials as they are biologically degradable.
In addition to their use as renewable raw materials in industrial production and manufacture, fructosyl polymers are attractive as food additives, for example as sweeteners. Polymers of various chain lengths are required for the various applications. While for the food sector polymers of short or medium chain length are preferred, technical applications, for example the production of surfactants, require polymers with a high degree of polymerization (DP).
Various processes have been described for producing fructan polysaccharides in plants by expressing fructosyltransferases of bacterial origin or for producing polyfructoses of medium chain length by expressing fructosyltransferases of plant origin. PCT/US89/02729, e.g., describes the possibility of generating carbohydrate polymers, particularly dextrane or polyfructose, in transgenic plants, namely specifically in the fruit of transgenic plants. In order to generate plants that are modified in that way it is proposed to use levan sucrose from microorganisms, particularly from Aerobacter levanicum, Streptococcus salivarius and Bacillus subtilis, or of dextrane sucrases from Leuconostoc mesenteroides. Neither the generation of the active enzyme, nor that of levan or dextrane nor the production of transgenic plants is described.
PCT/EP93/02110 discloses a method for producing transgenic plants which express the lsc gene of the levan sucrase from the gram-negative bacterium Erwinia amylovora. The plants produce a high-molecular, highly branched levan.
PCT/NL93/00279 describes the transformation of plants with chimeric genes that contain the sacB gene from Bacillus subtilis. Such plants produce a branched levan. The bacterial fructosyltransferases used in PCT/US89/02729, PCT/EP93/02110 and PCT/NL93/00279 synthesize levan, a β-2,6 linked fructosyl polymer which has numerous β-2,1 branchings. Due to the numerous branchings, however, levan involves decisive disadvantages for the technical processing and is therefore much less in demand as technical starting material than inulin which displays β-2,1 linkings. Presently, only one bacterial gene is known the gene product of which is involved in the synthesis of inulin, said gene being the ftf gene from Streptococcus mutans. PCT/NL93/00279 describes the transformation of plants with said gene which synthesize high-molecular inulin but in such small amounts that it cannot be economically utilized. PCT/EP97/02195, too, describes a process for producing transgenic, inulin-producing plants with the ftf gene from Streptococcus mutans. Like with the plants described in PCT/NL93/00279 the yield of high-molecular inulin is low. While it is possible to express the gene in plants if the gene was genetically engineered beforehand, the yield in inulin that can be obtained from transgenic plants is so low that the transgenic plants cannot be economically utilized.
Furthermore, PCT/NL96/00012 discloses DNA sequences which encode carbohydrate polymer-synthesizing enzymes as well as the production of transgenic plants using said DNA sequences. The sequences disclosed originate from Helianthus tuberosus. In accordance with PCT/NL96/00012 the sequences disclosed can be used to modify the fructan profile of petunia and potato but also of Helianthus tuberosus itself. Expression of the sequences disclosed which encode a sucrose-dependent sucrose fructosyltransferase (SST) or a fructan fructosyl transferase in transgenic plants allows the production of inulin. The average polymerization degree of the inulin is, however, DP=6 to DP=10. With such a polymerization degree the inulin cannot be considered long-chain. The process described in PCT/NL96/00012 does not allow to produce high-molecular inulin.
Recently, Rehm et al. (J. Bacteriology 180 (1998), 1305-1310) reported the generation of oligosaccharides in yeast by introducing an SST from Aspergillus foetidus. However, the polymerization degree of the product obtained was only DP=3.
DE 197 08 774.4 relates to the production of 1-kestose and nystose using enzymes having fructosyl polymerase activity. The tri- and tetrasaccharide may be produced in transgenic plants. The yield is high and in potato corresponds to the cellular content of sucrose. However, the production of longer-chain inulin is not described. The synthesis of polyfructoses by fungi is also discussed in many publications. Barthomeuf and Pourrat (Biotechnology Letters 17 (1995), 911-916), describe, e.g., an enzyme preparation of Penicillium rugulosum which has fructosyltransferase activity. The preparation exhibits various enzymatic properties and therefore does not represent a pure fructosyltransferase. DNA sequences of the fructosyltransferase gene are not known. Cairns et al. (New Phytologist 129 (1995), 299-308) describe a transient synthesis of tri-, tetra- and pentasaccharides from sucrose in the culture medium of Monographella nivalis. The underlying enzymatic activity appears to be of mainly hydrolytic nature since the polyfructoses are degraded again by the enzyme with increasing substrate exhaustion. Since no DNA sequence is known it is not possible to assess—relying on the homology with fructofuranosidases (invertases) as reference—whether a fructosyltransferase in the proper sense or an invertase is present.
It was shown for the fungus Aspergillus sydowi IAM 2544 that it is capable of generating polyfructoses of the inulin type. Harada et al. (in: Nishinari and Doi (Eds.), Food Hydrocolloids: Structures, Properties and Functions, Plenum, New York (1994), 77-82) describe, for example, the synthesis of inulin with conidia of Aspergillus sydowi. 125 g conidia were incubated in 25 1 20% sucrose solution. The product generated was purified by HPLC. However, such a process does not lend itself for a large-scale production of inulin. Maramatsu et al. (Agric. Biol. Chem. 52 (1988), 1303-1304) describe the production of fructooligosaccharides with mycelium of the same fungal strain (A. sydowi IAM 2544). The polymerization degree is reported to be 3 to 13. The enzymes involved in this process were not or only partially purified. Amino acid sequences or DNA sequences of the corresponding genes are not known. Instructions for the purification of the proteins are not or only incompletely given.